Method for Therapeutic Replenishment and Enrichment of Ocular Surface Lubrication

ABSTRACT

The present invention provides a pharmaceutical composition, and methods of use thereof, for treating ocular boundary deficiency, symptoms associated therewith, or undesired condition that is associated with or causes ocular boundary deficiency at the ocular surface. The pharmaceutical composition of the present invention comprises a human PRG4 protein, a lubricant fragment, homolog, or isoform thereof, suspended in an ophthalmically acceptable balanced salt solution. The pharmaceutical composition of the present invention may also comprise one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable demulcent, excipient, astringent, vasoconstrictor, emollient, sodium hyaluronate, hyaluronic acid, and surface active phospholipids, in a pharmaceutically acceptable carrier for topical administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/940,370filed on Nov. 5, 2010 which is a continuation of PCT Application No.PCT/US09/39887, filed Apr. 8, 2009, which claims priority benefit ofU.S. Provisional Application No. 61/051,112 filed May 7, 2008, each ofwhich is incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the management of ocular lubrication.In particular, the present invention relates to pharmaceuticalcompositions, and method of use thereof, for treating diseasesassociated with compromised lubrication at the corneal and conjunctivalsurfaces.

BACKGROUND

The proteoglycan 4 (prg4) gene encodes for highly glycosylated proteinstermed megakaryocyte stimulating factor (MSF), lubricin, and superficialzone protein (SZP) (1)). Lubricin was first isolated from synovial fluidand demonstrated lubricating ability in vitro similar to synovial fluidat a cartilage-glass interface (2). Lubricin was later identified as aproduct of synovial fibroblasts (3) and also shown to possess boundarylubricating ability at a latex-glass interface by Jay et al. (3-9).O-linked β(1-3)Gal-GalNAc oligosaccharides within a large mucin likedomain of 940 amino acids (10), encoded for by exon 6, were subsequentlyshown to mediate, in part, this boundary lubricating ability (8). SZPwas first localized at the surface of explant cartilage from thesuperficial zone and isolated from conditioned medium (11). SZP alsodemonstrated lubricating ability at a cartilage-glass interface (12).These molecules are collectively referred to as PRG4. PRG4 was alsoshown to be present at the surface of synovium (58), tendon (13), andmeniscus (14). In addition, PRG4 has been shown to contribute, both atphysiological and pathophysiological concentrations, to the boundarylubrication of apposing articular cartilage surfaces (59).

The functional importance of prg4 was shown by mutations that cause thecamptodactyly-arthropathy-coxa vara-pericarditis (CACP) disease syndromein humans. CACP is manifest by camptodactyly, noninflammatoryarthropathy, and hypertrophic synovitis, with coxa vara deformity,pericarditis, and pleural effusion (15). Also, in PRG4-null mice,cartilage deterioration and subsequent joint failure were observed (16).Therefore, PRG4 expression is a necessary component of healthy synovialjoints.

PRG4 is a member of the mucin family, which are generally abundant onepithelial linings and provide many functions, including lubrication andprotection from invading microorganisms (17). The functional propertiesof mucins are generally determined by specialized glycosylation patternsand their ability to form multimers through intermolecular disulfidebonds (18), both of which are altered in chronic diseases (e.g. cysticfibrosis, asthma) (17). Biochemical characterization of PRG4 isolatedfrom synovial fluid (2, 19) showed molecular heterogeneity inO-glycosylation, which appears to influence lubricating properties (8)Recently, PRG4 from bovine synovial fluid has been shown to exist asdisulfide-bonded dimers, in addition to the monomeric forms, assuggested by the conserved cysteine-rich domains at both N- andC-terminals, along with an unpaired cysteine at the C-terminal (20).

In tissues such as synovial joints, physicochemical modes of lubricationhave been classified as fluid film or boundary. The operativelubrication modes depend on the normal and tangential forces on thearticulating tissues, on the relative rate of tangential motion betweenthese surfaces, and on the time history of both loading and motion. Thefriction coefficient, μ, provides a quantitative measure, and is definedas the ratio of tangential friction force to the normal force. One typeof fluid-mediated lubrication mode is hydrostatic. At the onset ofloading and typically for a prolonged duration, the interstitial fluidwithin cartilage becomes pressurized, due to the biphasic nature of thetissue; fluid may also be forced into the asperities between articularsurfaces through a weeping mechanism. Pressurized interstitial fluid andtrapped lubricant pools may therefore contribute significantly to thebearing of normal load with little resistance to shear force,facilitating a very low μ. Also, at the onset of loading and/or motion,squeeze film, hydrodynamic, and elastohydrodynamic types of fluid filmlubrication occur, with pressurization, motion, and deformation actingto drive viscous lubricant from and/or through the gap between twosurfaces in relative motion.

The relevant extent to which fluid pressure/film versus boundarylubrication occurs classically depends on a number of factors (31). Whenlubricant film can flow between the conforming sliding surfaces, whichcan deform elastically, elastohydrodynamic lubrication occurs. Pressure,surface roughness, and relative sliding velocity determine when fullfluid lubrication begins to break down and the lubrication enters newregimes. As velocity decreases further, lubricant films adherent to thearticulating surfaces begin to contribute and a mixed regime oflubrication occurs. If the velocity decreases even further and only anultra-thin lubricant layer composed of a few molecules remain, boundarylubrication occurs. A boundary mode of lubrication is thereforeindicated by a friction coefficient (ratio of the measured frictionalforce between two contacting surfaces in relative motion to the appliednormal force) during steady sliding being invariant with factors thatinfluence formation of a fluid film, such as relative sliding velocityand axial load (35). For articular cartilage, it has been concludedboundary lubrication is certain to occur, although complemented by fluidpressurization and other mechanisms (36-39).

In boundary lubrication, load is supported by surface-to-surfacecontact, and the associated frictional properties are determined bylubricant surface molecules. This mode has been proposed to be importantbecause the opposing cartilage layers make contact over ˜10% of thetotal area, and this may be where most of the friction occurs (30).Furthermore, with increasing loading time and dissipation of hydrostaticpressure, lubricant-coated surfaces bear an increasingly higher portionof the load relative to pressurized fluid, and consequently, this modecan become increasingly dominant (31, 32). Boundary lubrication, inessence, mitigates stickslip (31), and is therefore manifest asdecreased resistance both to steady motion and the start-up of motion.The latter situation is relevant to load bearing articulating surfacesafter prolonged compressive loading (e.g., sitting or standing in vivo)(33). Typical wear patterns of cartilage surfaces (34) also suggest thatboundary lubrication of articular cartilage is critical to theprotection and maintenance of the articular surface structure.

With increasing loading time and dissipation of hydrostatic pressure,lubricant-coated surfaces bear an increasingly higher portion of theload relative to pressurized fluid, and consequently, μ can becomeincreasingly dominated by this mode of lubrication. A boundary mode oflubrication is indicated by values of μ during steady sliding beinginvariant with factors that influence formation of a fluid film, such asrelative sliding velocity and axial load. Boundary lubrication, inessence, mitigates stickslip, and is therefore manifest as decreasedresistance both to steady motion and the start-up of motion.

The accumulation of PRG4 within synovial fluid and at the articularsurface, are likely key functional determinants of PRG4's boundarylubricating ability. Recently, it was demonstrated that a significant,threefold secretion of PRG4 resulted from the dynamic shear loading ofcultured cartilage explants, as compared to free-swelling or staticallycompressed cultures (27). This PRG4 synthesis and secretion bychondrocytes could significantly contribute to the concentration of PRG4within synovial fluid, in both homeostatic and pathological conditionswhere physiological regulators are present (23). Although the amount ofPRG4 bound to the surface does not appear to correlate with secretionrates, previous studies suggest surface bound PRG4 can exchange withendogenous PRG4 in synovial fluid (25), especially under the influenceof mechanical perturbation (26, 27). Clarification of the spatial andtemporal aspects of PRG4 metabolism within the joint, particularly atthe articular surface, would further the understanding of PRG4'scontribution to the low-friction properties of articular cartilage, andpossibly lead to treatments to prevent loss of this function (40, 41).More remains to be determined about the processing, and the potentiallyadditional or alternative functions of various PRG4 molecules ofdifferent molecular weight (10, 27, 28, 61). Moreover, the combinationof chemical and mechanical factors to stimulate PRG4 expression inchondrocytes near the articular surface may be useful for creatingtissue engineered cartilage from isolated sub-populations (29) with asurface that is bioactive and functional in lubrication.

The precise mechanisms of boundary lubrication at biological interfacesare currently unknown. However, proteoglycan 4 (PRG4) may play acritical role as a boundary lubricant in articulating joints. Thissecreted glycoprotein is thought to protect cartilaginous surfacesagainst frictional forces, cell adhesion and protein deposition. Variousnative and recombinant lubricin proteins and isoforms have been isolatedand characterized. For instance, U.S. Pat. Nos. 5,326,558; 6,433,142;7,030223, and 7,361,738 disclose a family of human megakaryocytestimulating factors (MSFs) and pharmaceutical compositions containingone or more such MSFs for treating disease states or disorders, such asa deficiency of platelets. U.S. Pat. Nos. 6,960,562 and 6,743,774 alsodisclose a lubricating polypeptide, tribonectin, comprising asubstantially pure fragments of MSF, and methods of lubricating jointsor other tissues by administering tribonectin systemically or directlyto tissues.

SUMMARY OF THE INVENTION

The present invention provides, in various embodiments, pharmaceuticalcompositions, and methods of use thereof, for managing ocularlubrication, including the therapeutic replenishment and enrichment ofboundary lubricant molecules at the ocular surface. Described in certainembodiments of the present invention is the observation that PRG4 mRNAis expressed in human corneal and conjunctival epithelial cells, as wellas in mouse lacrimal and meibomian glands, indicating that PRG4 proteinis presented in these tissues on the ocular surface. Described incertain instances of the present invention is the observation that therole PRG4 protein serves on the ocular surface is to protect the corneaand conjunctiva against significant shear forces generated during aneyelid blink, contact lens wear, and other undesirable conditions. Theimpact of the tear film, including the impact of inflammation,proinflammatory cytokines, sex steroid imbalance and proteases on thecomposition and function of the films, suggest a course of therapy forocular tissues which promotes boundary lubrication.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for topical application to an ocular surfacecomprising a therapeutically effective concentration of a PRG4 proteinsuspended in an ophthalmically acceptable balanced salt solution. Thepharmaceutical composition of the present invention may also compriseone or more ophthalmically acceptable agents selected from the groupconsisting of an ophthalmically acceptable demulcent, ophthalmicallyacceptable excipient, ophthalmically acceptable astringent,ophthalmically acceptable vasoconstrictor, and ophthalmically acceptableemollient.

Exemplary ophthalmically acceptable demulcents contemplated in thepresent invention include, but are not limited to,carboxymethylcellulose sodium (e.g., about 0.2 to 2.5% w/v),hydroxyethyl cellulose (e.g., about 0.2 to 2.5% w/v), hypromellose(e.g., about 0.2 to 2.5% w/v), methylcellulose (e.g., about 0.2 to 2.5%w/v), dextran 70 (e.g., about 0.1% w/v), gelatin (e.g., about 0.01%w/v), glycerin (e.g., about 0.2 to 1% w/v), polyethylene glycol 300(e.g., about 0.2 to 1% w/v), polyethylene glycol 400 (e.g., about 0.2 to1% w/v), polysorbate 80 (e.g., about 0.2 to 1% w/v), propylene glycol(e.g., about 0.2 to 1% w/v), polyvinyl alcohol (e.g., about 0.1 to 4%w/v), povidone (e.g., about 0.1 to 2% w/v). Exemplary ophthalmicallyacceptable excipients/emollients contemplated in the present inventioninclude, but are not limited to, anhydrous lanolin (e.g., about 1 to 10%w/v), lanolin (e.g., about 1 to 10% w/v), light mineral oil (e.g.,≦about 50% w/v), mineral oil (e.g., ≦about 50% w/v), paraffin (e.g.,≦about 5% w/v), petrolatum (e.g., ≦about 100% w/v), white ointment(e.g., ≦about 100% w/v), white petrolatum (e.g., ≦about 100% w/v), whitewax (e.g., ≦about 5% w/v), yellow wax (e.g., ≦about 5% w/v). Anexemplary ophthalmically acceptable astringent contemplated in thepresent invention includes, but is not limited to, zinc sulfate (e.g.,about 0.25% w/v). Exemplary ophthalmically acceptable vasoconstrictorscontemplated in the present invention include, but are not limited to,ephedrine hydrochloride (e.g., about 0.123% w/v), naphazolinehydrochloride (e.g., about 0.01 to about 0.03% w/v), phenylephrinehydrochloride (e.g., about 0.08 to about 0.2% w/v), and tetrahydrozolinehydrochloride (e.g., about 0.01 to about 0.05% w/v).

In some of these embodiments, the demulcents, excipients, astringents,vasoconstrictors, emollients and electrolytes provide a means to deliverthe PRG4 protein in an ophthalmically acceptable manner. Ophthalmicallyacceptable compositions are suitable for topical application to theocular surface if they lack unacceptable eye toxicity, burning,itchiness, viscosity, blurred vision, etc. upon application.

In certain embodiments, the pharmaceutical composition of the presentinvention further comprises a therapeutically effective concentration ofone or more additional therapeutic agents, including but not limited to,sodium hyaluronate, hyaluronic acid, and phospholipid. Exemplaryphospholipid includes, but is not limited to,L-α-dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylethanolamine and sphingomyelin.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for topical application to an ocular surfacecomprising a therapeutically effective concentration of PRG4 proteinsuspended in an ophthalmically acceptable balanced salt solutioncomprising at least three electrolytes, including but not limited to,sodium chloride (NaCl) 0.64%, potassium chloride (KCl) 0.075%, calciumchloride dihydrate (CaCl2.2H2O) 0.048%, magnesium chloride hexahydrate(MgCl2.6H2O) 0.03%, sodium acetate trihydrate (C2H3NaO2.3H2O) 0.39%,sodium citrate dehydrate (C6H5Na3O7.2H2O) 0.17%, sodium hydroxide and/orhydrochloric acid (to adjust pH to approximately 7.5) with an osmolarityof approximately 300 mOsms/L.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for topical application to an ocular surfacecomprising a therapeutically effective concentration of PRG4 proteinsuspended in an ophthalmically acceptable balanced salt solution,comprised of sodium (Na+) of approximately 128 mM, potassium (K+) ofapproximately 24 mM, chloride (Cl−) of approximately 113 mM, calcium(Ca2+) of approximately 0.4 mM, magnesium (Mg2+) of approximately 0.3mM, HCO3− of approximately 5 mM, citrate of approximately 1 mM,phosphate of approximately 14 mM, acetate of approximately 15 mM, andsodium hydroxide and/or hydrochloric acid (to adjust pH to approximately7.5) with an osmolarity of approximately 300 mOsms/L.

The present invention further provides a method for treating ocularlubrication deficiency, or symptoms associated therewith, in anindividual in need. The method comprises topically administering to theocular surface of the individual in need a pharmaceutical compositioncomprising a therapeutically effective concentration of a PRG4 protein.In certain embodiments, the pharmaceutical composition comprising thePRG4 protein is administered in combination with an ophthalmicallyacceptable formulation comprising one or more ophthalmically acceptableagents selected from the group consisting of an ophthalmicallyacceptable demulcent, ophthalmically acceptable excipient,ophthalmically acceptable astringent, ophthalmically acceptablevasoconstrictor, and ophthalmically acceptable emollient.

In some embodiments, the pharmaceutical composition comprising the PRG4protein is administered in combination with an ophthalmically acceptablesolution comprising a therapeutically effective concentration of sodiumhyaluronate or hyaluronic acid, or a surface active phospholipid, asdiscussed above. In yet certain embodiments, the pharmaceuticalcomposition comprising the PRG4 protein is administered in combinationwith a phosphate buffered saline solution or an ophthalmicallyacceptable balanced salt solution comprising one or more electrolytes,as discussed above.

The present invention provides a method for treating a deficiency inocular lubrication or symptoms associated therewith, that due to tearloss or unstable tear film in the ocular boundary loop, such as androgendeficiency, Sjögren's syndrome and keratoconjunctivitis sicca (KCS).Such method comprises topically administering to the ocular surface of apatient in need the pharmaceutical composition of the present invention.

In certain embodiments, the present invention further provides a methodfor addressing and treating the conditions associated with unfavorableor deficient ocular lubrication. Exemplary conditions include, but arenot limited to aqueous or evaporative dry eye disease, Sjögren'ssyndrome, keratoconjunctivitis sicca, androgen deficiency, meibomiangland disease, estrogen replacement therapy, contact lens wear,refractive surgery, allergy, reduced tear film breakup time, allergy,ocular surface disorders, increased protease levels in the tear film andat the ocular surface, chronic inflammation, hyperosmolarity, and aging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents feedback loops within ocular surface boundarylubrication.

FIG. 2 illustrates PRG4 mRNA expression in human corneal epithelialcells. Human corneal epithelial cells were isolated from thecorneoscleral rims of male and female donors. Amplified samples werescreened for the presence of PRG4 products by using an Agilent 2100Bioanalyzer. Vertical lanes contain: L. MW ladder; 1. No templatecontrol; 2. Corneal tissue from a 33-year female; 4. Cultured cornealepithelial cells from a 70-year female; 6. Cultured corneal epithelialcells from a 53-year male.

FIG. 3 illustrates PRG4 mRNA expression in human conjunctival epithelialcells. Human corneal epithelial cells were isolated from thecorneoscleral rims of male and female donors. Amplified samples werescreened for the presence of PRG4 products by using agarose gelelectrophoresis. Vertical lanes contain: 1. MW ladder; 2. No templatecontrol; 4. Human female conjunctiva; 5. Human male conjunctiva.

FIG. 4 illustrates PRG4 mRNA expression in human corneoscleral rimtissue samples. L. Human corneal epithelial cells were isolated from thecorneoscleral rims of male and female donors. Amplified samples werescreened for the presence of PRG4 products by using an Agilent 2100Bioanalyzer. Vertical lanes contain: MW ladder; 1. Human liver cDNAstandard; 2. Corneoscleral rim tissue from a 24-year female; 3.Corneoscleral rim tissue from a 51-year female; 4. Human conjunctivalepithelial cells.

FIG. 5 illustrates PRG4 mRNA expression in human conjunctival impressioncytology samples. Conjunctival impression cytology samples were isolatedfrom male and female donors. Amplified samples were screened for thepresence of PRG4 products by using an Agilent 2100 Bioanalyzer. Verticallanes contain: L. MW ladder; 1-9. Conjunctival impression cytologysamples; 10. Repeat of human conjunctival epithelial cells (Lane 4 inFIG. 3).

FIG. 6 illustrates a friction test schematic. The corneal ocular surface(605) was fastened to the spherical end of an inert non-permeablesemi-rigid rubber plug cylinder (603) (radius r=6 mm). The plug cylinder(603) was attached to the rotational actuator of the mechanical testingmachine (Bose ELF 3200) forming the bottom articular surface. An annulus(601) (outer radius=3.2 mm, inner radius=1.5 mm) was punched from theeyelid (604). The annulus (601) was attached to the linear actuatorcoupled with an axial load (N) and torsion (τ) load cells, forming theupper articulating surface. Lubricant bath (602) was formed by securingan inert tube around the plug cylinder (603). ω is the angularfrequency.

FIG. 7 illustrates the reduction of in vitro lid/cornea kinetic frictionwith addition of PRG4 protein (lubricin).

FIG. 8 illustrates the reduction of in vitro lid/cornea kinetic frictionmeasured 1 minute after the addition of PRG4 protein (lubricin).

FIG. 9 illustrates the reduction of in vitro lid/cornea kinetic frictionmeasured 5 minutes after the addition of PRG4 protein (lubricin).

FIG. 10 illustrates the reduction of in vitro lid/cornea kineticfriction over time, following addition of PRG4 protein (lubricin).

DETAILED DESCRIPTION OF THE INVENTION

Provided in certain embodiments herein, is a method for treating ocularlubrication deficiency (e.g., ocular boundary lubrication deficiency),or symptoms associated therewith, in an individual in need thereofcomprising topically administering to the ocular surface of theindividual a pharmaceutical composition comprising a therapeuticallyeffective amount of PRG4 protein. Also provided in some embodimentsherein are pharmaceutical compositions comprising PRG4 protein in anophthalmically acceptable formulation. In specific embodiments, providedherein is a pharmaceutical composition suitable for topical applicationto an ocular surface comprising a therapeutically effective amount ofPRG4 suspended in an ophthalmically acceptable balanced salt solution,and may also be in combination with one or more ophthalmicallyacceptable agents selected from the group consisting of anophthalmically acceptable demulcent, an ophthalmically acceptableexcipient, an ophthalmically acceptable astringent, an ophthalmicallyacceptable vasoconstrictor, and an ophthalmically acceptable emollient.

Provided in some embodiments herein are pharmaceutical compositions, andmethods of use thereof, for treating a deficiency in ocular lubricationat the ocular surface (e.g., a deficiency of, such as decreased orundesirable, ocular boundary lubrication). A pharmaceutical compositionof certain embodiments of the present invention comprises an isolated orpurified PRG4 protein suspended in an ophthalmically acceptable balancedsalt solution in combination with one or more ophthalmic agents selectedfrom the group consisting of an ophthalmic demulcent, excipient,astringent, vasoconstructor, and emollient. In some embodiments, anypharmaceutical composition provided herein further comprises one or moreadditional therapeutic agents selected from the group consisting ofsodium hyaluronate, surface active phospholipids, and electrolytes in apharmaceutically acceptable carrier for topical administration.

The present invention provides, in certain embodiments, a novel approachto manage ocular lubrication, including the therapeutic replenishmentand enrichment of boundary lubricant molecules at the ocular surface. Itshould be noted that the importance and the mechanism of ocular boundarylubrication has not heretofore been recognized within the ophthalmiccommunity. For years, the scientific consensus within the orthopaedicresearch community was that hydrodynamic lubrication was by far thedominant mode of lubrication for articular cartilage, and that boundarylubrication was simply an afterthought. Moreover, those researchersstudying boundary lubrication at cartilage surfaces suggest thatboundary lubrication is likely only important under “high load and lowvelocity,” which are opposite to the conditions at the ocular surface,where there are relatively low axial loads and relatively fast slidingvelocities. See, e.g., (54). Moreover, boundary lubrication involvingthe corneal glyocalyx has not heretofore been considered. Jay et al.compared purified lubricating factor from bovine synovial fluid to“mucinous glycoprotein from human submandibular saliva and stimulatedtears,” and concluded “mucin secreted by the lacrimal gland did notlubricate,” overlooking the possibility that the corneal epithelium wasa source of lubricant or that boundary lubrication was an importantcontributor at the ocular surface. See, e.g., (55). The most recentmathematical models of tear film dynamics also ignore the possibility ofboundary lubrication, claiming a “lubrication approximation” for theheight of the tear film such that “the mucus layer on the cornea can betaken to provide a no-slip surface for the aqueous film” and that “itshould be noted that the model only predicts the evolution prior to the[tear film] thickness reaching some critically thin value at which themodel breaks down.” See, e.g., (57).

There is a need to manage ocular lubrication and protect the cornea andconjunctiva against significant shear forces generated from theundesirable conditions described herein, including, by way ofnon-limiting example, aqueous or evaporative dry eye disease, Sjögren'ssyndrome, keratoconjunctivitis sicca, androgen deficiency, meibomiangland disease, estrogen replacement therapy, contact lens wear,refractive surgery, allergy, reduced tear film breakup time, allergy,ocular surface disorders, increased protease levels in the tear film andat the ocular surface, chronic inflammation, hyperosmolarity, and aging.

In some instances, the loading of cornea and conjunctiva is likelydominated by shear forces. In certain instances, eyelid blinking, aswell as contact lens wear, generates significant stress upon ocularsurface epithelial cells, and this is especially true in the presence ofa compromised tear film. As shown in FIG. 1, it is suggested thatincreased shear stress leads to tear film instability, evaporative tearloss, hyperosmolarity, changes in swelling pressure and a feedbackelevation in shear stress. In some instances, increased shear stress isalso thought to promote inflammation, androgen deficiency and decreasedexpression of proteoglycans. In certain instances increased shear stressand its sequelae may, over time, lead to a loss of boundary lubricationat the ocular surface.

A deficiency in ocular lubrication and symptoms associated therewith canbe determine by any suitable method. In some instances, a deficiency inocular lubrication and symptoms associated therewith is defined eitherqualitatively (e.g., a feeling of low lubrication, dry eye, discomfort,etc.) or quantitatively (e.g., measured through mechanical, biochemical,electrical, optical or other methods of quantitative assays).

In certain instances, in undesirable conditions for ocular boundarylubrication, such those resulting from aqueous or evaporative dry eyedisease, Sjögren's syndrome, keratoconjunctivitis sicca, androgendeficiency, meibomian gland disease, estrogen replacement therapy,contact lens wear, refractive surgery, allergy, reduced tear filmbreakup time, allergy, ocular surface disorders, increased proteaselevels in the tear film and at the ocular surface, chronic inflammation,hyperosmolarity, and aging, a compromised tear film will exist. In someof these situations, increased evaporation may preclude efficient fluidfilm lubrication, but allow boundary lubrication and a molecularsacrificial mechanism to reduce shear stress at the cell surface.Certain embodiments of the present invention provide that therapeuticreplenishment and enrichment of boundary lubricant molecules at theocular surface would interrupt the feedback loop through which theunfavorable conditions associated with a deficiency in ocularlubrication promote ocular surface distress.

In certain instances, and as provided herein, PRG4 protein plays acritical role in the eye as a boundary lubricant. In some instances,this secreted glycoprotein protects the ocular surface to protect thecornea and conjunctiva against significant shear forces generated duringan eyelid blink, contact lens wear, and any other undesirable ocularboundary lubrication caused by chronic inflammation and hyperosmolaritythat result from dry eye disease, androgen deficiency, estrogenreplacement therapy, compromised tear film, allergy, aging, ocularsurface diseases, and increased protease levels in the tear film and atthe ocular surface. Given the relationship between osmotic pressure andthe electromechanical interactions within charged molecules, the presentinvention provides, in some embodiments, a pharmaceutical compositionfor managing a deficiency in ocular lubrication by modulatinghyperosmolarity or osmolarity at the ocular surface via interrupting thefeedback mechanisms that prevent secreted components from reducingfriction coefficients and mitigating shear stress.

In another exemplary embodiment, the present invention features asacrificial mechanism for ocular boundary lubrication, whereby surfacebound receptors reversibly bind one or more gel forming or surfactantconstructs. In some instances, the gel forming or surfactant constructsdetach during a shear event, thereby preventing the shear stress fromreaching (or reducing the shear stress reaching) the epithelial surface.In certain embodiments, following the transient shearing event, the gelforming and surfactant constructs, allowed to return to theirundisturbed equilibrium, rebind to the surface bound receptors. In someembodiments, the entire construct can detach during shear. One couldimagine, in certain instances, that the thermodynamics of thisequilibrium would increase the probability of release from the receptorwith increasing shear amplitude, but that any one association is easilyreversible.

In one embodiment of the current invention, the pharmaceuticalcomposition comprising a PRG4 protein suspended in an ophthalmicallyacceptable balanced solution is applied topically to the ocular surface,where the PRG4 protein associates or binds to. In certain instances ofthis embodiment, PRG4 acts as the surface bound receptor that is allowedto interact with endogenous proteins and proteoglycans within the tearfilm to establish a sacrificial mechanism to reduce the friction duringeyelid blinks at the ocular surface, prevent protein adsorption at theocular surface, and reduce dry spots caused by tear film instability.

In another embodiment of the current invention, PRG4 is appliedtopically and associates or binds to the ocular surface, in combinationwith one or more of hyaluronic acid and phospholipid constructs. Incertain instances of this embodiment, PRG4 acts as the surface boundreceptor that interacts with the exogenously supplied hyaluronic acidand/or phospholipids to establish the sacrificial mechanism to reducethe friction during eyelid blinks at the ocular surface, prevent proteinadsorption at the ocular surface, and reduce dry spots caused by tearfilm instability. In this embodiment, the hyaluronic acid andphospholipid constructs disassociate from the PRG4 during a shear event.In yet another embodiment, the entire construct detaches during a shearevent to prevent the shear stress from reaching the epithelium.

In yet another embodiment, functional fragments, multimers (e.g.,dimers, trimers, tetramers, etc.), homologs or orthologs of PRG4 act asthe surface receptor and/or gel forming constructs in the sacrificialmechanism. Functional fragments and homologs of PRG4 include those witha fewer repeats within the central mucin-like KEPAPTT-repeat (SEQ ID NO:4) domain, glycosylated and non-glycosylated forms of the protein,splice variants, recombinant forms, and the like. A lubricating fragmentof PRG4 exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%of the ophthalmic lubricating effect of human PRG4, as measuredqualitatively, mechanically, optically, electrically, or by biochemicalassay.

As used herein, the term “PRG4”, “PRG4 protein” or “proteoglycan 4”protein, is used interchangeably with the term “lubricin” protein. PRG4is used herein also to encompass the term megakaryocyte stimulatingfactor (MSF), that has been accepted for the UCL/HGNC/HUGO Human GeneNomenclature data base, and superficial zone protein (SZP). The PRG4 orlubricin protein as used herein refers to any isolated or purifiednative or recombinant lubricin proteins, homologs, functional fragmentsor motifs, isoforms, and/or mutants thereof. In certain embodiments, theisolated or purified PRG4 protein comprises an amino acid sequence for ahuman native or recombinant lubricin protein. In other embodiments, theisolated or purified PRG4 protein comprises an amino acid sequenceencoded by prg4gene exons that encode the full length PRG4 protein orisoforms' primary structures. The proteoglycan 4 (prg4) gene contains 12exons. The PRG4 protein used herein comprises an amino acid sequenceencoded by prg4gene exons 1-12, more preferably, exons 6-12, and mostpreferably, exons 9-12.

As used herein, the PRG4 protein includes any PRG4 proteins now known,or later described. In certain embodiments, a preferred PRG4 proteinamino acid sequence is provided in SEQ ID NO:1. The PRG4 protein sharesthe primary amino acid structure of any known PRG4 proteins or isoformswith at least 60% homology, preferably 75% homology, more preferably85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology. In certainembodiments, a preferred PRG4 protein has an average molar mass ofbetween 50 kDa and 400 kDa, comprising one or more biological activeportions of the PRG4 protein, or functional fragments, such as alubricating fragment, or a homolog thereof.

As used herein, the PRG4 protein comprises a biological active portionof the protein. As used herein, a “biologically active portion” of thePRG4 protein includes a functional fragment of a protein comprisingamino acid sequences sufficiently homologous to, or derived from, theamino acid sequence of the protein, which includes fewer amino acidsthan the full length protein, and exhibits at least one activity of thefull-length protein. Typically a biologically active portion comprises afunctional domain or motif with at least one activity of the protein. Abiologically active portion of a protein can be a polypeptide which is,for example, 10, 25, 50, 100, 200, or more amino acids in length. In oneembodiment, a biologically active portion of the PRG4 protein can beused as a therapeutic agent alone or in combination with othertherapeutic agents for treating undesirable or decreased ocular boundarylubrication.

The nucleic acid and amino acid sequences of several native andrecombinant PRG4 or lubricin proteins, and characterization of the PRG4proteins and various isoforms are disclosed in, for instance, U.S. Pat.Nos. 5,326,558; 6,433,142; 7,030,223; 7,361,738 to Turner et al., andU.S. Pat. Nos. 6,743,774 and 6,960,562 to Jay et al. U.S. PublicationNo. 20070191268 to Flannery et al. also discloses recombinant PRG4 orlubricin molecules useful in the present invention.

Methods for isolation, purification, and recombinant expression of aPRG4 protein are well known in the art. In certain embodiments, themethod starts with cloning and isolating mRNA and cDNA encoding PRG4proteins or isoforms using standard molecular biology techniques, suchas PCR or RT-PCR. The isolated cDNA encoding the PRG4 protein or isoformis then cloned into an expression vector, and further transformed andexpressed in a host cell for producing recombinant PRG4 protein.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant” alsoencompasses the ligation of nucleic acids having various coding regionsor domains or promoter sequences from different sources into anexpression cassette or vector for expression of, e.g., inducible orconstitutive expression of a fusion protein comprising an active domainof the PRG4 gene and a nucleic acid sequence amplified using a primer ofthe invention.

In certain embodiments, the PRG4 protein encoding nucleic acid maycontain one or more mutations, deletions, or insertions. In suchembodiments, the PRG4 protein encoding nucleic acid is at least 60%homology, preferably 75% homology, more preferably 85%, 90%, 95%, 96%,97%, 98%, 99%, or more homology, to a wild type PRG4 protein encodingnucleic acid.

As used herein, the term ‘cDNAs” includes DNA that is complementary tomRNA molecules present in a cell or organism mRNA that can be convenedinto cDNA with an enzyme such as reverse transcriptase. In certainembodiments, the cDNA encoding PRG4 protein is isolated from PRG4 mRNAexpressed in human corneal or conjunctival epithelial cells using anRT-PCR method well known in the art.

As used herein, the terms “polynucleotide,” “nucleic acid/nucleotide,”and “oligonucleotide” are used interchangeably, and include polymericforms of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides: agene or gene fragment, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, ribozymes, DNA, cDNA, genomic DNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. Polynucleotides may be naturally-occurring, synthetic,recombinant or any combination thereof.

A polynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also includes both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

As used herein, the term “polynucleotide sequence” is the alphabeticalrepresentation of a polynucleotide molecule. A polynucleotide iscomposed of a specific sequence of four nucleotide bases: adenine (A);cytosine (C); guanine (G); thymine (T); and uracil (U) in place ofthymine when the polynucleotide is RNA, instead of DNA. Thisalphabetical representation can be inputted into databases in a computerand used for bioinformatics applications such as, for example,functional genomics and homology searching.

As used herein, the term “isolated polynucleotide/cDNA” includespolynucleotide molecules which are separated from other polynucleotidemolecules which are present in the natural source of the polynucleotide.For example, with regard to genomic DNA, the term “isolated” includespolynucleotide molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”polynucleotide is free of sequences which naturally flank thepolynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide of interest) in the genomic DNA of the organism fromwhich the polynucleotide is derived. For example, in variousembodiments, the isolated polynucleotide molecule encoding the PRG4protein used in the invention can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the polynucleotide molecule in genomic DNA of the cell from whichthe polynucleotide is derived. Moreover, an “isolated” polynucleotidemolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, a “gene” includes a polynucleotide containing at leastone open reading frame that is capable of encoding a particularpolypeptide or protein after being transcribed and translated. Any ofthe polynucleotide sequences described herein may also be used toidentify larger fragments or full-length coding sequences of the genewith which they are associated. Methods of isolating larger fragmentsequences are known to those of skill in the art. As used herein, a“native or naturally-occurring” polynucleotide molecule includes, forexample, an RNA or DNA molecule having a nucleotide sequence that occursin nature (e.g., encodes a natural protein).

As used herein, the term “polypeptide” or “protein” is interchangeable,and includes a compound of two or more subunit amino acids, amino acidanalogs, or peptidomimetics. The subunits may be linked by peptidebonds. In another embodiment, the subunit may be linked by other bonds,e.g., ester, ether, etc. As used herein, the term “amino acid” includeseither natural and/or unnatural or synthetic amino acids, includingglycine and both the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlyreferred to as an oligopeptide. Peptide chains of greater than three ormore amino acids are referred to as a polypeptide or a protein.

In certain embodiments, the PRG4 protein used herein refers to PRG4proteins or various homologs or isoforms thereof, that are naturally orrecombinantly expressed in humans or other host cells. As used herein,“express” or “expression” includes the process by which polynucleotidesare transcribed into RNA and/or translated into polypeptides. If thepolynucleotide is derived from genomic DNA, expression may includesplicing of the RNA, if an appropriate eukaryotic host is selected.Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors canbe obtained commercially or assembled by the sequences described inmethods well known in the art, for example, the methods described belowfor constructing vectors in general. As used herein, the term “vector”includes a self-replicating nucleic acid molecule that transfers aninserted polynucleotide into and/or between host cells. The term isintended to include vectors that function primarily for insertion of anucleic acid molecule into a cell, replication vectors that functionprimarily for the replication of nucleic acid and expression vectorsthat function for transcription and/or translation of the DNA or RNA.Also intended are vectors that provide more than one of the abovefunction.

As used herein, a “host cell” is intended to include any individual cellor cell culture which can be, or has been, a recipient for vectors orfor the incorporation of exogenous polynucleotides and/or polypeptides.It is also intended to include progeny of a single cell. The progeny maynot necessarily be completely identical (in morphology or in genomic ortotal DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. The cells may be prokaryotic oreukaryotic, and include but are not limited to bacterial cells, yeastcells, insect cells, animal cells, and mammalian cells, including butnot limited to murine, rat, simian or human cells. As used herein, a“host cell” also includes genetically modified cells. The term“genetically modified cells” includes cells containing and/or expressinga foreign or exogenous gene or polynucleotide sequence which in turnmodifies the genotype or phenotype of the cell or its progeny.“Genetically modified” also includes a cell containing or expressing agene or polynucleotide sequence which has been introduced into the cell.For example, in this embodiment, a genetically modified cell has hadintroduced a gene which gene is also endogenous to the cell. The term“genetically modified” also includes any addition, deletion, ordisruption to a cell's endogenous nucleotides. As used herein, a “hostcell” can be any cells that express a human PRG4 protein.

As used herein, “homologs” are defined herein as two nucleic acids orpeptides that have similar, or substantially identical, nucleic acids oramino acid sequences, respectively. The term “homolog” furtherencompasses nucleic acid molecules that differ from one of thenucleotide sequences due to degeneracy of the genetic code and thusencodes the same amino acid sequences. In one of the preferredembodiments, homologs include allelic variants, orthologs, paralogs,agonists, and antagonists of nucleic acids encoding the PRG4 protein(e.g., SEQ ID NO:1).

As used herein, the term “orthologs” refers to two nucleic acids fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode peptides having the same orsimilar functions. In particular, orthologs of the invention willgenerally exhibit at least 80-85%, more preferably 85-90% or 90-95%, andmost preferably 95%, 96%, 97%, 98%, or even 99% identity, or 100%sequence identity, with all or part of the amino acid sequence of anyknown PRG4 proteins (e.g., SEQ ID NO:1), isoforms, or analogs thereof,and will exhibit a function similar to these peptides. As also usedherein, the term “paralogs” refers to two nucleic acids that are relatedby duplication within a genome. Paralogs usually have differentfunctions, but these functions may be related.

To determine the percent sequence identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of one polypeptide for optimalalignment with the other polypeptide or nucleic acid). The amino acidresidues at corresponding amino acid positions are then compared. When aposition in one sequence is occupied by the same amino acid residue asthe corresponding position in the other sequence, then the molecules areidentical at that position. The same type of comparison can be madebetween two nucleic acid sequences. The percent sequence identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., percent sequenceidentity=numbers of identical positions/total numbers of positions×100).Preferably, the isolated amino acid homologs included in the presentinvention are at least about 50-60%, preferably at least about 60-70%,and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or moreidentical to an entire amino acid sequence of any known PRG4 protein(e.g., SEQ ID NO:1).

In certain embodiments, an isolated nucleic acid homolog encoding thePRG4 protein comprises a nucleotide sequence which is at least about40-60%, preferably at least about 60-70%, more preferably at least about70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and even more preferably atleast about 95%, 96%, 97%, 98%, 99%, or more identical to a nucleotidesequence encoding amino acid sequences of such PRG4 protein (e.g., SEQID NO:1).

The determination of the percent sequence identity between two nucleicacid or peptide sequences is well known in the art. For instance, theVector NTI 6.0 (PC) software package (InforMax, Bethesda, Md.) todetermine the percent sequence identity between two nucleic acid orpeptide sequences can be used. In this method, a gap opening penalty of15 and a gap extension penalty of 6.66 are used for determining thepercent identity of two nucleic acids. A gap opening penalty of 10 and agap extension penalty of 0.1 are used for determining the percentidentity of two polypeptides. All other parameters are set at thedefault settings. For purposes of a multiple alignment (Clustal Walgorithm), the gap opening penalty is 10, and the gap extension penaltyis 0.05 with blosum62 matrix. It is to be understood that for thepurposes of determining sequence identity when comparing a DNA sequenceto an RNA sequence, a thymidine nucleotide is equivalent to a uracilnucleotide.

Furthermore, the PRG4 protein used herein includes PRG4 protein encodedby a polynucleotide that hybridizes to the polynucleotide encoding PRG4protein under stringent conditions. As used herein, “hybridization”includes a reaction in which one or more polynucleotides react to form acomplex that is stabilized via hydrogen bonding between the bases of thenucleotide residues. The hydrogen bonding may occur by Watson-Crick basepairing, Hoogstein binding, or in any other sequence-specific manner.The complex may comprise two strands forming a duplex structure, threeor more strands forming a multi-stranded complex, a singleself-hybridizing strand, or any combination of these. A hybridizationreaction may constitute a step in a more extensive process, such as theinitiation of a PCR reaction, or the enzymatic cleavage of apolynucleotide by a ribozyme.

Hybridization reactions can be performed under different stringentconditions. The present invention includes polynucleotides capable ofhybridizing under reduced stringency conditions, more preferablystringent conditions, and most preferably highly stringent conditions,to polynucleotides encoding PRG4 protein described herein. As usedherein, the term “stringent conditions” refers to hybridizationovernight at 60° C. in 10×Denhart's solution, 6×SSC, 0.5% SDS, and 100mg/ml denatured salmon sperm DNA. Blots are washed sequentially at 62°C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1%SDS, and finally 0.1×SSC/0.1% SDS. As also used herein, in certainembodiments, the phrase “stringent conditions” refers to hybridizationin a 6×SSC solution at 65° C. In other embodiments, “highly stringentconditions” refer to hybridization overnight at 65° C. in 10×Denhart'ssolution, 6×SSC, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA.Blots are washed sequentially at 65° C. for 30 minutes each time in3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1%SDS. Methods for nucleic acid hybridizations are well known in the art.Accordingly, the PRG4 proteins encoded by nucleic acids used hereininclude nucleic acid having at least 60% homology, preferably 75%homology, more preferably 85%, more preferably 90%, most preferably 95%,96%, 97%, 98%, 99% homology to a polynucleotide sequence that encodes ahuman PRG4 protein (e.g., SEQ ID NO:1) or a specific isoform or homologthereof.

Moreover, the PRG4 proteins used herein can also be chimeric protein orfusion protein. As used herein, a “chimeric protein” or “fusion protein”comprises a first polypeptide operatively linked to a secondpolypeptide. Chimeric proteins may optionally comprise a third, fourthor fifth or other polypeptide operatively linked to a first or secondpolypeptide. Chimeric proteins may comprise two or more differentpolypeptides. Chimeric proteins may comprise multiple copies of the samepolypeptide. Chimeric proteins may also comprise one or more mutationsin one or more of the polypeptides. Methods for making chimeric proteinsare well known in the art. In certain embodiments of the presentinvention, the chimeric protein is a chimera of PRG4 protein with otherPRG4 protein isoforms.

As used herein, an “isolated” or “purified” protein, polynucleotide ormolecule means removed from the environment in which they naturallyoccur, or substantially free of cellular material, such as othercontaminating proteins from the cell or tissue source from which theprotein polynucleotide or molecule is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations separated from cellular components of the cells from whichit is isolated or recombinantly produced or synthesized. In certainembodiments, the language “substantially free of cellular material”includes preparations of a PRG4 protein having less than about 30% (bydry weight) of other proteins (also referred to herein as a“contaminating protein”), more preferably less than about 20%, stillmore preferably less than about 10%, and most preferably less than about5% of other proteins. When the protein or polynucleotide isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the preparation of the protein of interest.

In certain embodiments, the present invention provides a pharmaceuticalcomposition suitable for topical administration to an ocular surface ofan individual in need a pharmaceutically effective concentration of PRG4protein suspended in an ophthalmically acceptable balanced saltsolution, and in combination with one or more ophthalmically acceptableagents. The ophthalmically acceptable agents can be selected from thegroup consisting of an ophthalmically acceptable demulcent, excipient,astringent, vasoconstrictor, and emollient. As used herein, the term“effective concentration or amount” or “therapeutically effectiveconcentration or amount” is intended to mean a nontoxic but sufficientconcentration or amount of a PRG4 protein or other therapeutic agents toprovide the desired therapeutic effects. The concentration or amountthat is effective will vary from subject to subject, depending on theage and general condition of the individual, the particular agents, andthe like. Thus, it is not always possible to specify an exact effectiveconcentration or amount. However, an appropriate effective concentrationor amount in any individual case may be determined by one of ordinaryskill in the art using routine experimentation. Furthermore, the exacteffective concentration or amount of a PRG4 protein and othertherapeutic agent incorporated into a composition or dosage form of thepresent invention is not critical, so long as the concentration iswithin a range sufficient to permit ready application of the solution orformulation so as to deliver an amount of the PRG4 protein and otheractive agents that is within a therapeutically effective range.

In certain embodiments, the pharmaceutically effective concentration ofPRG4 protein is in a range of 10-10,000 μg/mL, preferably 50-5,00 mg/mL,and more preferably 100-300 mg/mL. As used herein, the ophthalmicallyacceptable agents comprising the ophthalmically acceptable demulcents,excipients, astringents, vasoconstrictors, and emollients that are fullydefined in the Code of Federal Regulations 21CFR349.

As used herein, the term “topical administration” is used in itsconventional sense to mean delivery of the composition comprising thePRG4 protein and one or more ophthalmically acceptable agents to theeye. In general, topical administration is achieved through a liquidformulation for eye drops or lavage and provides a local effect.

In certain embodiments, any pharmaceutical composition described hereincomprise or the aforementioned ophthalmically acceptable agents are orcan be combined with one or more of carboxymethylcellulose sodium (e.g.,about 0.2 to about 2.5% w/v), hydroxyethyl cellulose (e.g., about 0.2 toabout 2.5% w/v), hypromellose (e.g., about 0.2 to about 2.5% w/v),methylcellulose (e.g., about 0.2 to about 2.5% w/v), dextran 70 (e.g.,about 0.1% w/v), gelatin (e.g., about 0.01% w/v), glycerin (e.g., about0.2 to about 1% w/v), polyethylene glycol 300 (e.g., about 0.2 to about1% w/v), polyethylene glycol 400 (e.g., about 0.2 to about 1% w/v),polysorbate 80 (e.g., about 0.2 to about 1% w/v), propylene glycol(e.g., about 0.2 to about 1% w/v), polyvinyl alcohol (e.g., about 0.1 toabout 4% w/v), povidone (e.g., about 0.1 to about 2% w/v), zinc sulfate(e.g., about 0.25% w/v), anhydrous lanolin (e.g., about 1 to about 10%w/v), lanolin (e.g., about 1 to about 10% w/v), light mineral oil (e.g.,≦about 50% w/v), mineral oil (e.g., ≦about 50% w/v), paraffin (e.g.,≦about 5% w/v), petrolatum (e.g., ≦about 100% w/v), white ointment(e.g., ≦about 100% w/v), white petrolatum (e.g., ≦about 100% w/v), whitewax (e.g., ≦about 5% w/v), yellow wax (e.g., ≦about 5% w/v), ephedrinehydrochloride (e.g., about 0.123% w/v), naphazoline hydrochloride (e.g.,about 0.01 to about 0.03% w/v), phenylephrine hydrochloride (e.g., about0.08 to about 0.2% w/v), and tetrahydrozoline hydrochloride (e.g., about0.01 to about 0.05% w/v). In certain instances, percent amounts utilizedherein are percent amounts by weight.

In further embodiments, the pharmaceutical composition of the presentinvention comprising a PRG4 protein in combination with one or moreophthalmically acceptable agents discussed above further comprises atherapeutically effective concentration of hyaluronic acid or sodiumhyaluronate in the range of 10-100,000 μg/mL, preferably 500-5,000mg/mL. Furthermore, the pharmaceutical composition of the presentinvention further comprises one or more surface active phospholipids inthe range of 10-10,000 μg/mL, such surface active phospholipids include,but are not limited to, L-α-dipalmitoylphosphatidylcholine (DPPC),phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (Sp), or other neutral and polar lipids.

The pharmaceutical composition of the present invention may furthercomprise one or more pharmaceutically acceptable carriers or vehiclescomprising any acceptable materials, and/or any one or more additivesknown in the art. As used herein, the term “carriers” or “vehicle” referto carrier materials suitable for topical drug administration. Carriersand vehicles useful herein include any such materials known in the art,which are nontoxic and do not interact with other components of thecomposition in a deleterious manner. Various additives, known to thoseskilled in the art, may be included in the composition. For example,solvents, including relatively small amounts of alcohol, may be used tosolubilize certain drug substances. Other optional additives includeopacifiers, antioxidants, fragrance, colorant, gelling agents,thickening agents, stabilizers, surfactants, and the like. Other agentsmay also be added, such as antimicrobial agents, to prevent spoilageupon storage, i.e., to inhibit growth of microbes such as yeasts andmolds. Suitable antimicrobial agents are typically selected from thegroup consisting of the methyl and propyl esters of p-hydroxybenzoicacid (i.e., methyl and propyl paraben), sodium benzoate, sorbic acid,imidurea, and combinations thereof. Permeation enhancers and/orirritation-mitigating additives may also be included in thepharmaceutical composition of the present invention.

In certain embodiments, the pharmaceutical composition of the presentinvention is prepared in a pharmaceutically acceptable carrier, such asa phosphate buffered saline or an osmotically balanced salt solution oftear electrolytes, including one or more of sodium chloride in about 44%to about 54% mole fraction, potassium chloride in about 8% to about 14%mole fraction, sodium bicarbonate in about 8% to about 18% molefraction, potassium bicarbonate in about 0% to about 4% mole fraction,calcium chloride in about 0% to about 4% mole fraction, magnesiumchloride in about 0% to about 4% mole fraction, trisodium citrate inabout 0% to about 4% mole fraction, and hydrochloric acid in about 0% toabout 20% mole fraction or sodium hydroxide in about 0% to about 20%mole fraction. In certain embodiments, the pharmaceutical carrier can beformulated to generate an aqueous electrolyte solution in about 150-200mM range. Other suitable formulations, such as ointments, creams, gels,pastes, and the like, suitable for topical administration, are alsocontemplated in the present invention. In certain embodiments,electrolytes provide proper osmotic balance when combined with PRG4 tomake a solution ophthalmically acceptable.

The present invention further provides a method for treating decreasedor undesired ocular boundary lubrication, symptoms associated therewith,or a condition that is associated with or causes a deficiency in ocularlubrication, in an individual in need thereof, comprising topicallyadministering to the ocular surface of the individual in need apharmaceutical composition comprising a therapeutically effective amountof PRG4 protein. In one embodiment, the method of the present inventioncomprises topically administering a pharmaceutical compositioncomprising the therapeutically effective amount of the PRG4 protein thatis suspended in a phosphate buffered saline solution or anophthalmically acceptable balanced salt solution comprising one or moreelectrolytes. In yet other embodiment, the method of the presentinvention comprising topically administering a pharmaceuticalcomposition comprising the PRG4 protein formulated in an ophthalmicallyacceptable formulation comprising one or more additional ophthalmicallyacceptable agent as discussed above.

As used herein, the term “treating or treatment” refers to reduction inseverity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. The term“treating or treatment” also encompasses both prevention of a disorderin a predisposed individual and treatment of the disorder in aclinically symptomatic individual.

In certain embodiments, the decreased ocular boundary lubrication iscaused by increased evaporative tear loss or unstable tear film in theocular boundary loop. Such decreased or undesired ocular boundarylubrication is associated with aqueous or evaporative dry eye disease,Sjögren's syndrome, keratoconjunctivitis sicca (KCS), androgendeficiency, meibomian gland disease, estrogen replacement therapy,contact lens wear, refractive surgery, allergy, reduced tear filmbreakup time, compromised tear film, ocular surface disorders, increasedprotease levels in the tear film and at the ocular surface, chronicinflammation, hyperosmolarity, and aging. As discussed above, theincreased shear stress leads to tear film instability, evaporative tearloss, hyperosmolarity, changes in swelling pressure and a feedbackelevation in shear stress. Increased shear stress also promotesinflammation, androgen deficiency and decreased expression ofproteoglycans. Over time, increased shear stress and its sequelae leadsto a loss of boundary lubrication at the ocular surface. Accordingly,the present invention provides a method for reducing shear stress byreplenishing and enriching the expression of proteoglycans, such as PRG4protein at the ocular surface, so as to prevent or increase ocularboundary lubrication.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims. These and many other variations and embodiments of the inventionwill be apparent to one of skill in the art upon a review of theappended description and examples.

EXAMPLES Example 1 PRG4 mRNA Expression in Human Corneal andConjunctival Epithelial Cells

Human corneal epithelial cells were isolated from the corneoscleral rimsof male and female donors. Cells were processed either directly (n=8),or first cultured in phenol red-free keratinocyte serum free media(n=2). Bulbar conjunctivae (n=2), conjunctival impression cytologysamples (n=9), immortalized human conjunctival epithelial cells afterculture (n=1), NOD mouse lacrimal glands (n=5 adult mice/sex, 10glands/sample), and BALB/c mouse meibomian glands (n=7 adult mice/sex,glands from 28 lids/sample) were obtained during surgical procedures.These samples were processed for the analysis of PRG4 mRNA by usingprimarily RT-PCR (n=18 human, all mouse) and Affymetrix GeneChips (n=4human corneas). The PRG4 primers for PCR spanned over 1 kbp of intronsequences, in order to suppress amplification of contaminatingchromosomal DNA (Table 1). Amplified samples were screened for thepresence of PRG4 products by using agarose gel electrophoresis and anAgilent 2100 Bioanalyzer. To confirm the identity of amplicons, PCRproducts from cornea samples (n=2), conjunctival epithelial cells (n=1)and a human liver standard (n=1) were sequenced with a 3100 GeneticAnalyzer at the Massachusetts Eye and Ear Infirmary DNA SequencingCenter for Vision Research (Boston, Mass.) and resulting data wereanalyzed with BLASTn searches of GenBank databases.

TABLE 1 Oligonucleotide primers designed for RT-PCR analysis of PRG4 mRNA Orien- Nucleotide Amplicon Species tationsequence (5′-3′) Exons Size (bp) Human Sense GATGCAGGGTACCCCAAA  9-12526 (SEQ ID NO: 2) Anti- CAGACTTTGGATAAGGTC  sense TGCC(SEQ ID NO: 3)

It was demonstrated that PRG4 mRNA is present in all human corneal andconjunctival epithelial cell and impression cytology samples. Theidentity of PRG4 PCR products was confirmed by DNA sequence analysis(Table 2). The results show that PRG4 is transcribed in human cornealand conjunctival epithelial cells.

TABLE 2 Identification of amplicon sequences from human cornea,conjunctival and liver samples Aligned Base Total Base Sequencing PairsTo Pairs from BLASTn Search Direction Human PRG4 Amplicon Identity HumanLiver Standard A Forward 495 500 Human PRG4 A Reverse 488 491 Human PRG4B Forward 496 499 Human PRG4 B Reverse 498 500 Human PRG4 Human Cornea(24 year old female) A Forward 497 499 Human PRG4 A Reverse 490 492Human PRG4 B Forward 500 504 Human PRG4 B Reverse 498 501 Human PRG4Human Cornea (51 year old female) A Forward 498 499 Human PRG4 A Reverse474 489 Human PRG4 B Forward 496 498 Human PRG4 B Reverse 490 491 HumanPRG4 Human Conjunctival Epithelial Cells A Forward 496 499 Human PRG4 AReverse 490 492 Human PRG4 B Forward 495 499 Human PRG4 B Reverse 474491 Human PRG4 Two different samples (A & B) of each preparation weresequenced in forward and reverse directions. The human cornea sampleswere epithelial cells from the corneoscleral rims of female donors. Thegene accession number for human PRG4 is NM_005807.

Example 2 Reduction of Friction In Vitro with the Addition of PRG4(Lubricin)

An in vitro friction test with clinically relevant interfaces, such asan ocular surface-eyelid and ocular surface-contact lens interface isdescribed below. Clinically relevant methods capable of quantitativelyassessing the lubricating ability of artificial tears are currentlylacking. Friction tests with synthetic (e.g. latex and glass) ornon-ocular ‘native’ surfaces (e.g. umbilical cord vein segments) mayfacilitate some, but likely not all of the molecular interactions thatoccur during articulation/blinking. Indeed, the relevance of dataobtained with non-tissue interfaces is unclear.

An annulus-on-disk rotational test configuration has been shown to beideal for studying boundary lubrication at an articularcartilage-cartilage interface. A boundary mode of lubrication isindicated by kinetic friction being invariant with factors thatinfluence formation of a fluid film, including sliding velocity andaxial load. This is because surface-to-surface contact is occurring, andsurface bound molecules contribute to lubrication (by decreasingfriction and wear). Boundary lubrication has been discovered to be acritical and operative mechanism at the ocular surface, like it is atthe articular cartilage surface. Therefore, the in vitro friction testpreviously developed and characterized to study boundary lubrication atan articular cartilage-cartilage interface was modified for the study ofocular surface-eye lid and ocular surface-contact lens interfaces.

To determine the test conditions in which boundary lubrication isdominant at the ocular surface-eyelid and ocular surface-contact lensinterfaces, the dependence of frictional properties on axial load andsliding velocity was examined. Normal fresh human ocular surfaces(resected corneas with ˜3 mm of sclera) were obtained from the Lions EyeBank of Alberta. The resected corneas were stored in Optisol-GS at 4° C.and used within 2 weeks. Eyelids (age 60-80 years old) were obtainedfrom the University of Calgary Body Donation Program within 1-3 daysafter death and used immediately or stored at −20° C. in saline for atmost 2 weeks until use. Comparative lubricants consisted of Lens PlusSterile Saline Solution (Advanced Medical Optics) as a negative control;Systane® Lubricant Eye Drops (Alcon Laboratories), Refresh TearsLubricant Eye Drops (Allergan), Aquify® Long Lasting Comfort Drops (CIBAVision) and Blink® Tears Lubricant Eye Drops (Advanced Medical Optics)as test lubricants.

The friction test schematic is shown in FIG. 6. The corneal ocularsurface (605) was fastened to the spherical end of an inertnon-permeable semi-rigid rubber plug cylinder (603) (radius r=6 mm) byapplying super glue to the sclera. This plug cylinder (603) was attachedto the rotational actuator of the mechanical testing machine (BoseELF3200) thus forming the bottom articular surface. An annulus (601) (outerradius=3.2 mm, inner radius=1.5 mm) was punched from the eyelid (604),and was attached to the linear actuator coupled with an axial load (N)and torsion (τ) load cell, thus forming the upper articulating surface.Lubricant bath 602 was formed by securing an inert tube around the plugcylinder (603).

Samples were first tested in saline, then in one of the three (3) testlubricants. The lubricant bath was filled with ˜0.3 ml, and thearticulating surfaces allowed to equilibrate with the lubricant. Thesample surfaces were slowly (0.05 mm/s) brought into contact andcompressed until the spherical plug flattened out and the entire annulareyelid surface was in contact with the cornea (605). The resultingnormal stress (calculated from axial load as, in units of MPa, asN/(π[r² _(outer)−r² _(inner)]) can be varied by using differentstiffness rubber plugs to mimic physiological stresses ˜5 kPa. The testsequence was initiated by preconditioning the sample by rotating +4revolutions (rev) and reset with −4 revolutions at a physiologicallyrelevant effective linear sliding velocity, veff=30 mm/s (whereveff=ωReff, ω is the angular frequency, and Reff=2.4 mm is the effectiveradius calculated by integrating the shear stress distribution over theannular contact area). Samples were then tested by rotating +4revolutions, immediately followed by −4 reset revolutions at veff=30,10, 1, 0.3 and then 30 mm/s, with a dwell time of 12 second between eachrevolution. The test sequence was then be repeated in the oppositedirection of rotation.

To evaluate the lubrication properties of the ocular surface, twofriction coefficients (μ) of the form μ=τ/(R_(eff)N)) where is torque,R_(eff) is effective radius, and N is axial load, described above. Astatic friction coefficient, which reflects the resistance to the onsetof motion, μ_(static) was calculated as the peak value of μ, just after(within ˜10°) the start of rotation. An average kinetic frictioncoefficient, which reflects the resistance to steady state motion,<μ_(kinetic)> was calculated from μ averaged during the third and fourthcomplete test revolution. Both μ_(static) and <μ_(kinetic)> wereaveraged for the + and − revolutions in each test to account forpotential directional effects on τ measurements. Data was collected at afrequency of 20 Hz.

The results of lubricin (PRG4) added to the corneal surface at aconcentration in the range of 100-300 ug/mL are shown in FIG. 7.Lubricin had a friction lowering effect at the eyelid interface, both interms of kinetic and static friction, at all velocities. At aconcentration 1/10th of that of physiological hyaluronic acid, lubricinwas similar to Blink® Tears Lubricant Eye Drops, which containshyaluronic acid. In combination, the two lubricants are better thaneither alone.

FIG. 8 demonstrates the reduction of in vitro cornea/lid kineticfriction measured during the first minute after the addition oflubricin, as compared to Aquify® eye drops. Lubricants were thoroughlywashed from the ocular surface using saline between tests. A synergisticeffect (reduced μ_(kinetic) over either alone) was evident when Aquify®(with hyaluronic acid) was combined with lubricin. The saline repeat waslower than the original saline control. This showed a retention oflubricin's effect even after washing with saline, suggesting that themolecules were binding to the ocular surface, and that lubricindemonstrated superior retention time as compared to sodium hyaluronatealone.

FIG. 9 demonstrates the reduction of in vitro cornea/lid kineticfriction measured during the 5th minute after the addition of lubricin,as compared to Aquify® eye drops. A synergistic effect (reducedμ_(kinetic) over either alone) was evident when Aquify® (with hyaluronicacid) was combined with lubricin. The friction coefficient of Aquify®had returned to statistical equivalence to saline after 5 minutes,whereas lubricin remains lower, as did the combination of lubricin andhyaluronic acid.

FIG. 10 shows the reduction of kinetic friction coefficient over time,following addition of lubricin. Again, the continual reduction suggestedbinding to the ocular surface.

Example 3 Treatment of Deficient Ocular Boundary Lubrication In Vivo

A patient complaining of ocular surface irritation is examined forocular lubrication or conditions associated with a deficiency in ocularlubrication by measuring symptoms greater than 2 positive responses onthe McMonnies questionnaire, greater than a score of 5 on the OcularSurface Disease Index (OSDI), or through evidence of some symptoms onthe Visual Analog Scale, in combination with objective signs includingone or more of a reduced tear film breakup time (less than ≈10 seconds),inferior lateral tear meniscus osmolarity greater than 308 mOsms/L, lowSchirmer strip value (less than ≈10 mm), sodium fluorescein corneal orconjunctival staining (scores >0 with multiple macropunctates),significant debris resulting from impression cytology, meibomian glanddysfunction however determined, a decrease in the rate of post-blinkdisplacement of a contact lens, a change in the spatiotemporal transferfunction of a contact lens following application of a series of pressureimpulses, a decrease in the rate of post-blink interferometric tear filmrelaxation, an increase in the concentration of proinflammatorycytokines, a reduced concentration of lactoferrin or lysozyme, or anincrease in the rate of post-blink point spread function decoherence.

The patient administers 1 to 2 drops on the surface of each eye asolution containing 200 μg/mL PGR4 protein suspended in anophthalmically acceptable balanced salt solution. The patient isinstructed to close their eyes for 10 seconds.

Follow-up visits may track a reduction in inferior lateral tearosmolarity, increased tear film breakup time, or the otheraforementioned signs. In particular if the tear film osmolarity isreduced from an abnormal value (perhaps 330 mOsms/L) to a more normalvalue (perhaps 304 mOsms/L), the therapeutic modulation andreplenishment of the ocular surface lubrication would be deemedsuccessful.

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SEQUENCE LIST SEQ ID NO: 1MAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIRKIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPVVDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGAEGETPHMLLRPHVFMPEVTPDMDYLPRVPNQGIIINPMLSDETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNVVTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVW YNCP SEQ ID NO: 2: GATGCAGGGTACCCCAAA (human, sense) SEQ ID NO: 3: CAGACTTTGGATAAGGTCTGCC (human, antisense)

1. A method for treating dry eye disease, or symptoms associatedtherewith, in an individual in need thereof comprising topicallyadministering to the ocular surface of the individual a pharmaceuticalcomposition comprising an ophthalmically acceptable therapeuticallyeffective concentration of PRG4.
 2. The method of claim 1 comprisingtopically administering the composition together with one or moreophthalmically acceptable agents selected from the group consisting of ademulcent, an excipient, an astringent, a vasoconstrictor, an emollient,and an electrolyte salt.
 3. The method of claim 1 wherein thepharmaceutical composition comprising the PRG4 is administered togetherwith an ophthalmically acceptable solution of sodium hyaluronate orhyaluronic acid.
 4. The method of claim 3 wherein the hyaluronate saltor acid is present at a concentration of 10-100,000 μg/mL.
 5. The methodof claim 3 wherein the hyaluronate salt or acid is present at aconcentration of 500-5,000 μg/mL.
 6. The method of claim 1 comprisingadministering the composition together with an ophthalmically acceptablesurface active phospholipid selected from the group consisting ofL-α-dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylethanolamine and sphingomyelin.
 7. The method of claim 6comprising administering the phospholipid at a concentration of10-10,000 μg/mL.
 8. The method of claim 1 comprising administering thecomposition together with a phosphate buffered saline solutioncomprising at least sodium phosphate and sodium chloride.
 9. The methodof claim 1 comprising administering the composition together with anophthalmically acceptable balanced salt solution comprising one or moreelectrolytes selected from the group consisting of potassium chloride,sodium bicarbonate, potassium bicarbonate, calcium chloride, magnesiumchloride, a sodium citrate, hydrochloric acid, and sodium hydroxide. 10.The method of claim 1 comprising administering recombinant PRG4 proteinor functional fragment thereof.
 11. The method of claim 1 comprisingadministering a purified naturally occurring PRG4 protein.
 12. Themethod of claim 1 comprising administering the composition together withan ophthalmically acceptable solution comprising hyaluronate salt orhyaluronic acid and a surface active phospholipid selected from thegroup consisting of L-α-dipalmitoylphosphatidylcholine,phosphatidylcholine, phosphatidylethanolamine and sphingomyelin.
 13. Themethod of claim 1, wherein the individual is further in need oftreatment for Sjogren's syndrome, keratoconjunctivitis sicca, androgendeficiency, meibomian gland disease, estrogen replacement therapy,contact lens wear, refractive surgery, allergy, reduced tear filmbreakup time, compromised tear film, ocular surface disorders, increasedprotease levels in the tear film and at the ocular surface, chronicinflammation, hyperosmolarity, aging, or a combination thereof.
 14. Amethod for treating ocular discomfort comprising topically administeringto the ocular surface a pharmaceutical composition comprising PRG4 in anophthalmically acceptable vehicle at a concentration effective to reducetear film osmolarity.
 15. The method of claim 14 wherein the compositionis administered together with one or more ophthalmically acceptableagents selected from the group consisting of a demulcent, an excipient,an astringent, a vasoconstrictor, an emollient, an electrolyte, ahyaluronate salt or hyaluronic acid, and a surface active phospholipid.16. The method of claim 14 wherein the discomfort is caused by acondition selected from the group consisting of aqueous or evaporativedry eye disease, Sjogren's syndrome, keratoconjunctivitis sicca,androgen deficiency, meibomian gland disease, estrogen replacementtherapy, contact lens wear, refractive surgery, allergy, reduced tearfilm breakup time, compromised tear film, allergy, ocular surfacedisorders, increased protease levels in the tear film and at the ocularsurface, chronic inflammation, hyperosmolarity, aging, and combinationsthereof.
 17. The method of claim 1 wherein the composition comprises aconcentration of PRG4 effective to reduce tear film osmolarity.
 18. Themethod of claim 1 wherein the composition comprises a concentration ofPRG4 of 10-10,000 μg/mL.
 19. The method of claim 1 wherein thecomposition comprises a concentration of PRG4 of 50-500 μg/mL.
 20. Themethod of claim 1 wherein the composition comprises a concentration ofPRG4 of 100-300 μg/mL.
 21. The method of claim 14 wherein thecomposition comprises a concentration of PRG4 effective to treat dry eyedisease.
 22. The method of claim 14 wherein the composition comprises aconcentration of PRG4 of 10-10,000 μg/mL.
 23. The method of claim 14wherein the composition comprises a concentration of PRG4 of 50-500μg/mL.
 24. The method of claim 14 wherein the composition comprises aconcentration of PRG4 of 100-300 μg/mL.